Antigen-specific major histocompatibility complex (MHC)-restricted T-cell responses are an important component of immune responses against viral infections and tumors. Design and development of immunotherapy intervention depends upon understanding the target antigen as well as its capability to be efficiently presented to T-cells along with MHC class I and class II molecules.
With modern techniques, it is possible to determine the affinity of target antigen peptide to the antigen binding groove of MHC class I and class II molecules. However, it lo is not an easy task to predict the immunogenicity of a given antigenic peptide in the outbred human population, given our varying T-cell repertoires. The complexity is further increased with certain tumor antigens which are often recognized as xe2x80x9cselfxe2x80x9d peptides. Thus, a gene encoding a tumor antigen will be expressed in normal autologous cells without any change in nucleotide sequence.
Many adenocarcinomas, such as breast, ovarian, pancreatic, and colorectal, are highly expressed on the cell surface and secrete abnormal (underglycosylated) MUC-1 mucin. As a result of underglycosylation, MUC-1 mucin on these adenocarcinomas has exposed peptide epitopes. Hull, et al., (1989) Cancer Commun. 1:261-267; Burchell et al., (1987) Cancer Res. 47:5476-5482. This contrasts with normal ductal epithelium cells, where MUC-1 mucin is expressed on the apical surface and has a peptide core of a conserved tandem repeat of 20 amino acid units that is highly glycosylated and therefore has a hidden (cryptic) peptide core. In this normal situation, it is believed that the antigenic regions of MUC-1 are immunologically shielded.
Peptide epitopes on the tandem repeat regions of the MUC-1 mucin peptide core have been recognized as potential target antigens for immunotherapy of certain adenocarcinomas. Gendler et al., (1988) J. Biol. Chem. 263:12820-12823; Siddiqui et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2320-2323; Longenecker, et al., (1993) Immunologists. 1:89-95. It has been demonstrated that MUC-1 peptide specific T-cells have the potential to kill MUC-1 mucin bearing tumor cells. Agrawal et al., (1996) J. Immunol. 156:2089-2095. The following permissive peptide epitopes have also been defined: (1) epitope of the MUC-1 peptide-core for class II restricted CD4+ T-cell response and (2) an epitope which has the capability to bind to HLA.A11, HLA.A2.1, HLA.A3 and HLA.A1 Agrawal et al., (1995) Cancer Res. 55:2257-2261; Domenech et al., (1995) J. Immunol. 155:4766-4774. The usefulness of these peptides as potential vaccine candidates for the inmmunotherapy of various cancers depends upon their ability to generate strong CD4+ and CD8+ T-cell responses. It is generally feasible to determine the immunogenicity of a target peptide in mice after in vivo priming.
It has been reported (Agrawal et al., J. Immunol. and Agrawal et al., Cancer Res., supra) that MUC-1 antigen peptide specific CD4+ and CD8+ T-cells were isolated from PBLs obtained from healthy multiparous donors but not from nulliparous women or from men. However, in those studies, subjects were primed in vivo, and isolated T-cells were stimulated in vitro with soluble MUC-1 antigen peptide as antigen.
Recent research suggests that a primary CD8+ cytotoxic T-cell lymphocyte (CTL) response can be generated in vitro by stimulation of T-cells with mutant T2 or RMA-S cell lines that were treated, or xe2x80x9cloaded,xe2x80x9d with peptide. DeBruijn et al., (1992) Eur. J. Immunol. 21:2963-2970; DeBruijn et al., (1992) Eur. J. Immunol. 22:3013-3020; Stauss et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7871-7875; Houbiers et al., (1993) Eur. J. Immunol. 23:2072-2077. As used in this specification, a liposome that has been xe2x80x9cloadedxe2x80x9d with peptide is a formulated product with either membrane-associated and/or intravesicular peptide antigen. Such a xe2x80x9cloaded liposomexe2x80x9d is used as a delivery vehicle to xe2x80x9cloadxe2x80x9d cells with peptide antigen. Thus, a xe2x80x9cloaded cellxe2x80x9d is one that has effectively received, or taken up, peptide antigen. A loaded antigen-presenting cell (APC) is one that has taken up peptide antigen and expresses the antigen at the cell surface in the context of MHC class I or class II molecules. In addition, it was shown that antigen specific CTL could be generated in vitro using murine spleen cells having a high concentration of exogenous peptide. Alexander et al., (1991) J. Exp. Med. 173:849-858; Carbone et al., (1988) J. Exp. Med. 167:1767-1779.
Exogenously provided soluble peptides generally go through the endo-lysosomal presentation pathway for presentation in context of MHC class II molecules. Townsend et al., (1989) Annu. Rev. Imununol. 7:601-624; Unanue et al., (1987) Science 236:551-557. pH insensitive liposomes were shown to sensitize the APCs for class II restricted presentation. Furthermore, it has been shown that at high concentration of encapsulated antigen peptide, a pH insensitive liposome can deliver antigen to both endocytic and cytoplasmic locations for presentation by both MHC class I and MHC class II molecules. Harding et al., (1991) J. Immunol. 147:2860-2863; Zhou et al., (1994) Immunomethods 4:229-235.
PBLs pulsed with soluble peptides have been shown to be incapable of inducing primary T-cells in vitro. Germain et al., (1993) Annu. Rev. Immunol. 11:403-450. It was also shown that liposome encapsulated antigen was efficiently presented by DC but not macrophages to stimulate primary CTLs. Nair et al., (1993) J. Virol, 67:4062-4069. The purification and isolation of dendritic cells (DCs) is however, a difficult task and requires a large number of PBLs or bone marrow stem cells.
Dendritic cells were initially considered to be potential APCs to prime naive T-cells. Steinman, (1991) Annu. Rev. Immunol. 9:271-296. Dendritic cells have been used as APCs for in vitro stimulation of primary antigen-specific CTL responses (DeBrujin et al., Eur. J. Immunol. 22, supra, Nair et al., supra); Macatonia et al., (1989) J. Exp. Med. 169:1255-1264; Macatonia et al., (1991) Immunology. 74:399-406; Mehta-Damani et al., (1994) J. Immunol. 153:996-1003; Nair et al., (1992) J. Exp. Med. 175:609-612. It has been suggested that DCs are capable of intensive aggregation with unprimed T-cells and express a high density of accessory molecules, such as B7.1 and B7.2. Such accessory molecules are critical for stimulation of naive resting T-cells (Steinman, supra). B7.1 is one the xe2x80x9csecond signalxe2x80x9d receptors referred to as co-stimulatory molecules. It is the ligand for CD28 and is critical for the induction of TH1 responses. B7.2 is also a CD28 ligand and is associated with TH2 responsiveness. Also included in the category of co-stimulatory molecules is ICAM-1, which is the natural ligand of LFA, but is also shown to bind to MUC-1. Reginbald et al., (1996) Cancer Res. 56:4244.
However, DCs are not good candidates for (1) determining the immunogenicity of various peptides for immunotherapy and (2) stimulation of T-cells for expansion for adoptive cell therapy. In this regard, the prior art relates to generation of antigen-specific CD8+ CTL responses using DCs. The prior art does not suggest how to generate antigen-specific CD4+ CTL responses. The skilled artisan will recognize that CD4+ cytotoxic T-cells exist that will via class II-restricted peptide presentation. In addition the art does not suggest how to generate a mixture of antigen-specific T-cells that are CD8+ (T-cytotoxic) and CD4+ (T-helper).
The present invention provides a method for generating activated T-cells, comprising:
(a) combining liposome-encapsulated peptide antigen with a plurality of peripheral blood lymphocytes to produce antigen-loaded antigen-presenting cells;
(b) combining naive or anergic T-cells with said antigen-loaded antigen-presenting cells;
(c) isolating activated T-cells from the combination of step (b).
In a further embodiment, the present invention provides such a method wherein said activated T-cells are T helper cells and provides a method wherein said activated T-cells are cytotoxic T-cells.
In a still further embodiment, the invention provides such a method, wherein said liposome comprises monophosphoryl lipid A.
In yet another embodiment, the invention provides such a method, wherein said peptide antigen is BLP-25.
The invention also includes such a method wherein the combination of step (b) comprises IL-7 and IL-12.
The invention further includes such a method wherein said activated T-cell comprises a CD4 receptor and a method wherein said activated T-cell comprises a CD8 receptor.
In yet another embodiment; the invention comprises such a method wherein said activated T-cell is antigen-specific.
In other embodiments, the invention comprises such a method, wherein said antigen is MUC-1, or wherein said antigen is BLP-25.
In another embodiment, the present invention provides a method for producing a cellular vaccine, comprising combining liposome-encapsulated peptide antigen with a plurality of peripheral blood lymphocytes to produce antigen-loaded antigen-presenting cells, which comprise a cellular vaccine.
The present invention also provides a method for treating a patient suffering from cancer, comprising treating said patient with a pharmaceutically effective amount of a cellular vaccine, wherein said vaccine is produced by combining a plurality of peripheral blood lymphocytes with liposome-encapsulated peptide antigen to produce antigen-loaded antigen-presenting cells.